Specific genetic modification of the activity of trehalose-6-phosphate synthase and expression in a homologous or heterologous environment

ABSTRACT

A method for the preparation of a eukaryotic organism, for example selected from plants, animals and fungi, showing constitutive, inducible and/or organ specific expression of a specifically modified TPS gene, which comprises the steps of providing a TPS gene; designing a suitable modification to the TPS gene by aligning the gene with the corresponding gene of yeast and establishing which part of the gene extends beyond the 5′ terminus of the yeast gene; deleting or inactivating a part of the N-terminal region of the TPS gene extending beyond the 5′ terminus of the yeast gene, in order to achieve an increased trehalose-6-phosphate synthase activity; cloning the thus modified gene into an expression vector under the control of a constitutive, inducible and/or organ-specific promoter; transforming a plant cell or tissue with the thus obtained expression vector; and regenerating a complete plant from the transformed plant cell or tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/065,184, filed Feb. 24, 2005 (now U.S. Pat. No. 7,781,646), which isa continuation of U.S. patent application Ser. No. 10/110,502, filedApr. 15, 2002 (now U.S. Pat. No. 6,872,870), which filed under 35 U.S.C.§371 from international application No. PCT/EP99/07913, filed on Oct.15, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for obtaining eukaryoticorganisms, i.e. plants, animals or fungi, with elevated activity and/oraltered regulatory capacity of trehalose-6-phosphate synthase. Theinvention also relates to specifically modified alleles of thetrehalose-6-phosphate synthase genes which display unexpected changes incatalytic activity and/or regulatory capacity, and to the transformedplants, or other eukaryotic organisms containing these constructs. Theinvention is also related to novel methods to measure the level oftrehalose-6-phosphate and the activity of trehalose-6-phosphatesynthase.

2. Description of the Related Art

The biosynthesis of trehalose consists of two enzymatic steps catalyzedby trehalose-6-phosphate synthase (TPS), which synthesizestrehalose-6-phosphate, and by trehalose-6-phosphate phosphatase (TPP),which forms trehalose. The genes of the trehalose metabolism have beendiscovered first in yeast and in bacteria, organisms that were known fora long time to accumulate trehalose. Recently, homologues of these geneshave also been found in higher plants and animals in which appreciablelevels of trehalose had never been detected. However, up to now it hasnot been possible to demonstrate enzymatic trehalose-6-phosphatesynthase activity of these TPS gene products in any in vitro system.Their expression in heterologous systems also does not result in hightrehalose accumulation. No successful usage of these plant or animal TPSgenes to improve commercially important properties in homologous orheterologous systems has been reported.

In addition to its classical role in storage sugar accumulation,trehalose metabolism is known to play important roles in stressresistance, control of glucose influx into glycolysis andglucose-induced signalling. As outlined below, these phenotypicproperties are of high industrial importance.

An amazing capacity for adaptation to survival under strong or evencomplete dehydration is present in yeast cells, fungal spores, certaininvertebrate species and resurrection plants, which resume their vitalfunctions as soon as they are again in contact with water. Theseanhydrobiotic organisms also withstand freezing, strong vacuum, highdoses of ionizing radiation, high pressure and extreme temperatureswithout suffering damage and many of them accumulate the non-reducingdisaccharide trehalose as a protein and membrane protectant.

The protectant function of trehalose has also been demonstrated invitro. Addition of trehalose to cells, organelles, enzymes, antibodiesand foods preserves them under total dehydration for long periods. Italso protects them against a variety of other stress conditions, such ashigh temperature, high pressure and freezing.

In vascular plants, very few species are known where the presence oftrehalose has been demonstrated in a convincing way. However, in theso-called desert resurrection plant Selaginella lepidophylla a hightrehalose level is present. This plant is able to withstand successfullycomplete dehydration, as opposed to all other higher plants includingcrop plants.

Deletion mutants in the TPS gene in bacteria and yeast are unable tosynthesize trehalose and they lose osmotolerance, thermotolerance andtolerance to high pressure. This suggests that the TPS gene is involvedin various forms of tolerance.

It would be highly desirable to be able to express trehalose-6-phosphatesynthase activity in plants, animals, micro-organisms or specific partsthereof in order to render them tolerant to stress. In this way, cropplants could be cultured in regions suffering occasionally orcontinuously from heat, drought or freezing. Perishable foods from plantor animal origin could be preserved by simple dehydration, enablingstorage over a prolonged period of time and transport over longdistances.

SUMMARY OF THE INVENTION

The present invention for the first time allows to obtain hightrehalose-6-phosphate synthase activity and high accumulation oftrehalose in organisms where trehalose is normally not made or notaccumulated to appreciable levels, such as most higher plants andanimals. Hence, it allows for the first time highly-efficient andcontrolled use of trehalose accumulation in higher plants and animals toenhance stress resistance.

This is achieved in the invention by means of a method for thepreparation of an eukaryotic organism, for example selected from plants,animals and fungi, showing constitutive, inducible and/or organ specificexpression of a specifically modified TPS gene, comprising the steps of:

a) providing a TPS gene;

b) designing a suitable modification to the TPS gene by aligning thegene with the corresponding gene of yeast and establishing which part ofthe gene extends beyond the 5′ terminus of the yeast gene;

c) deleting or inactivating a part of the N-terminal region of the TPSgene extending beyond the 5′ terminus of the yeast gene, preferably thecomplete extending part thereof, in order to achieve an increasedtrehalose-6-phosphate synthase activity of the gene;

d) cloning the thus modified gene into an expression vector under thecontrol of a constitutive, inducible and/or organ-specific promoter;

e) transforming a plant cell or tissue with the thus obtained expressionvector; and

f) regenerating a complete plant from the transformed plant cell ortissue.

The inactivation of the part of the N-terminal region of the TPS geneextending beyond the 5′ terminus of the yeast gene can be accomplishedby mutagenesis.

It has been found according to the invention that truncation of variousgenes originating from plants can increase their functionality whenexpressed in yeast. An increased accumulation of trehalose and hightrehalose-6-phosphate synthase activity in comparison to the untruncatedgene was observed. By using a constitutive, inducible or organ-specificpromoter the expression can be modified and controlled in differentways. The induction can be tissue specific, for example for fruits, timespecific, or induced by changes in the environmental conditions. In thelatter category heat induction, drought induction, etc. can be included.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural diagram of the expression vector pBN35;

FIG. 2 is a structural diagram of plasmid pIBT101;

FIG. 3 is a structural diagram of plasmid pIBT102;

FIG. 4 is a structural diagram of plasmid pIBT103;

FIG. 5 is a structural diagram of plasmid pIBT104;

FIG. 6 is a representation of the growth on glucose and fructosecontaining medium of Wild Type and tps1Δ strains transformed with ayeast expression vector containing a complete or the truncated S.lepidophylla TPS gene;

FIG. 7 is a structural diagram of pCVV1;

FIG. 8 is a graph of absorbance of Tre6P standards with respect toconcentration;

FIG. 9 is a schematic representation of the method used to make taggedversions of TPS genes;

FIG. 10 is a structural diagram of plasmids containing TPS genes;

FIG. 11 is an illustration of western blot results using anti-HAantibodies;

FIG. 12 is an illustration of results for PVD164 and PVD165;

FIG. 13 is a diagram of the structures of chimaeric fusions of TPS andTPP proteins;

FIG. 14 is a graph of concentrations of glycolytic metabolites withrespect to time;

FIG. 15 is a graph of concentrations of metabolite concentrationsmeasured during exponential growth and stationary phase;

FIG. 16 is an overview of the trehalose-6-phosphate assay;

FIG. 17 is a bar graph showing a comparison of specific phosphotrehalaseactivity in strains EcVV1 and EcVV3; and

FIG. 18 is a bar graph showing the results of trehalose-6-phosphatemeasured in extracts from collected yeast cells.

The functionality of the modified TPS gene for inferring thermotolerancecan be checked in the following test system. Trehalose is required forthe acquisition of thermotolerance in yeast. The tps1Δ and tps1Δ tps2Δyeast deletion mutants are thermosensitive and the phenotype is restoredby complementation with the corresponding homologous gene. To determinewhether plant or animal TPS is functionally similar to yeast TPS1, tps1Δand tps1Δ tps2Δ yeast mutants transformed with a plasmid harboring thedesired gene are tested for the acquisition of thermotolerance. Theability of the cells to acquire thermotolerance is measured by a lethalheat-shock.

In yeast trehalose metabolism is essential for growth on glucose.Deletion of the TPS gene causes uncontrolled influx of glucose intoglycolysis resulting in hyperaccumulation of sugar phosphates and lossof free phosphate and ATP. Trehalose-6-phosphate is known to inhibithexokinase activity in vitro and the TPS enzyme therefore is thought toexert this control also in vivo by restricting hexokinase activity.

Glucose-induced signalling, and also signaling directly or indirectlyinduced by related sugars, such as sucrose, plays an important role inmany organisms for proper reaction to the availability of externalsugar, such as in yeast, or to the internal production of sugar, such asin photosynthetic plants or in the digestive system of animals. In yeastthe presence of external glucose, or related sugars, triggers severalsignalling pathways which causes rapid adaptation of metabolism to themaximal production of ethanol and to rapid growth. In photosyntheticplants sugar-induced signalling controls photosynthetic activity andpartitioning of the sugar between the source (photosynthetic parts) andsink organs (non-photosynthetic parts, in particular roots, seeds andfruits). In animals sugar-induced signalling controls the rate ofabsorption of the sugar from the blood by the storage organs, forinstance in mammals it controls the rate of absorption of the bloodsugar glucose by the liver.

TPS mutants of yeast are deficient in glucose-induced signalling, adeficiency that is thought to be due to the absence oftrehalose-6-phosphate inhibition of hexokinase activity. In higherplants, where trehalose is not accumulated in appreciable amounts, thefunction of the trehalose metabolism genes is not well understood.Plants in which heterologous TPS or TPP genes have been expresseddisplay altered photosynthetic activity and source-sink partitioning ofsugar which indicates possible effects on sugar-induced signallingpathways. This is thought to be due to changes in thetrehalose-6-phosphate level caused by the expression of the TPS or TPPgenes (Patent application Zeneca-Mogen 6047 PCT).

In the present invention, the unexpected situation is demonstrated thattruncation of the N-terminal part of plant TPS genes increases theircatalytic activity and therefore likely their regulatory capacity.Hence, the present invention allows alteration of sugar-inducedsignalling in a more efficient way in higher plants and possiblyanimals. Moreover, it allows to achieve this by homologous geneticmodification in principle in whatever plant and animal species, i.e. bythe expression of a truncated form of the homologous TPS enzyme.

In the present invention this alteration in sugar-induced signalling isachieved by the same steps as described hereinabove for the improvementof stress resistance.

The functionality of the modified genes for restoration of the controlof glucose influx into glycolysis and restoration of glucose-inducedsignalling can be checked in the following test system. TPS mutants ofyeast are not capable of growing on glucose because they are defectivein control of glucose influx into glycolysis and glucose-inducedsignalling. It is shown according to the invention that expression ofmodified TPS genes in the tps1Δ yeast strain restores growth on glucose,indicating restoration of the appropriate glucose-signalling controlsrequired for growth.

The present invention according to a further aspect thereof provides fora novel method for the measurement of trehalose-6-phosphate andtrehalose-6-phosphate synthase. This method is a highly reliable methodfor the quantitative determination of trehalose-6-phosphate. The levelof trehalose-6-phosphate in all organisms where it has been measured upto now is very low, in the range of 100-200 μM. This low concentrationmakes an accurate quantification by classical methods, for instance byHPLC, difficult and not very reliable. Chromatographic methods are alsotedious because the samples can only be measured one at a time. Otherresearch groups have used the inhibition of yeast hexokinase bytrehalose-6-phosphate as an indirect way to estimate the concentrationof trehalose-6-phosphate present in cell extracts (Blazquez M. A.,Lagunas R., Gancedo C. and Gancedo J. M., 1993, FEBS Lett. 329, 51-54).However, in general such assays are easily prone to interference withother compounds present in the cell extract, especially when derivedfrom organisms like plants and animals which contain many compounds notpresent in yeast.

In the present invention precise, quantitative measurement of thetrehalose-6-phosphate level is achieved by a novel enzymatic assay whichmakes use of the purified phosphotrehalase enzyme, preferably fromBacillus subtilis. The method comprises the following steps:

a) extraction of cells to be analyzed with an extraction medium,preferably a strong acid, that destroys all enzymatic activity but doesnot degrade trehalose-6-phosphate;

b) neutralization of the extract

c) centrifugation of the extract;

d) separation of the acidic compounds, including trehalose-6-phosphate,present in the supernatant from alkaline and neutral compounds,preferably by means of an anion exchange column;

e) treatment of the fraction containing the acidic compounds withpurified phosphotrehalase from Bacillus subtilis to degradetrehalose-6-phosphate quantitatively into glucose-6-phosphate andglucose;

f) separation of the glucose produced in step e) from theglucose-6-phosphate produced and from the remaining sugar phosphatespresent, preferably by means of a second anion exchange column;

g) determination of the glucose present, preferably by means of aglucose oxidase and peroxidase assay.

The glucose level measured is identical to the level oftrehalose-6-phosphate originally present in the cell extract.

The method established for the measurement of trehalose-6-phosphate canbe extended to the measurement of trehalose-6-phosphate synthaseactivity. Up to now there is no method available that allows themeasurement of trehalose-6-phosphate synthase activity based directly onthe rate of trehalose-6-phosphate formation. Trehalose-6-phosphatesynthase catalyzes the synthesis of trehalose-6-phosphate and UDP fromthe substrates glucose-6-phosphate and UDP-Glucose.

The classical method for determination of trehalose-6-phosphate synthaseactivity that is universally used measures the formation of the secondproduct of the enzyme, UDP (Hottiger, T., Schmutz, P., and Wiemken, A.,1987, J. Bacteriol. 169: 5518-5522). However, in cell extracts otherenzymes, e.g. glycogen synthase, are present that are able to produceUDP. Therefore, this method is prone to interference from otherenzymatic reactions. A method that directly measures the formation oftrehalose-6-phosphate is much more preferable. In addition, the saidmethod does not allow the continuous measurement of enzyme activity as afunction of time, since termination of the reaction is necessary beforeUDP can be measured.

It is now shown according to the invention that usage of the purifiedphosphotrehalase enzyme allows to achieve both goals at once. For thispurpose a coupled assay has been developed in whichtrehalose-6-phosphate is directly and continuously converted to glucoseby means of purified phosphotrehalase and the glucose producedcontinuously measured using the glucose oxidase/peroxidase method. Inthis assay phosphotrehalase, glucose oxidase and peroxidase are presentin excess whereas the trehalose-6-phosphate synthase in the cell extractis the limiting factor in the formation of the coloured product.

The present invention further relates to plants and other eukaryoticorganisms that show constitutive, inducible and/or organ specificexpression of a specifically modified TPS gene, which plants or othereukaryotic organisms are obtainable by means of the method of theinvention. The invention further relates to seeds of those plants orvegetatively reproducible structures of those plants, such as cuttings,somatic embryo's, protoplasts, as well as the further generations ofprogeny derived from those seeds and structures. The invention alsorelates to the further progeny of the other eukaryotic organisms.

The TPS gene can be derived from various sources, such as plants, inparticular Selaginella lepidophylla, Arabidopsis thaliana, rice, apple,sugar beet, sunflower (Helianthus annuus), tobacco (Nicotiana tabacum),soybean (Glycine max). The various genes can be expressed in homologousand heterologous environments.

The eukaryotic organism to be transformed can thus be a plant, eitherthe plant from which the gene is derived and now so modified that amodification in the activity of the TPS activity is obtained, or aheterologous plant. Especially preferred hosts for the modified gene arecrop plants, in particular plants that are not inherently stressresistant, but can be made stress resistant by the method of theinvention. As alternative the goal of the modification can be increasedphotosynthetic productivity and/or improved carbon partitioning in thewhole plant or in specific plant parts.

Other eukaryotic organisms to be transformed are fungi and animals oranimal cells. Examples of fungi for which an increase in TPS activitymay be beneficial are Aspergillus niger, Agaricus bisporus, Pichiapastoris, Kluyveromyces lactic and methylotrophic yeasts. An example ofa yeast is Saccharomyces cerevisiae. Animal cells are for examplemammalian and invertebrate cell cultures used for production of proteinsand small molecules, invertebrate cells used for baculovirus expression.

The present invention will be further illustrated in the followingexamples, which are not intended to be limiting.

EXAMPLES General Materials and Methods

Reagents

Baker or Sigma reagents of analytical grade were used. The restrictionand modification enzymes were from Boehringer-Mannheim. The ZAP cDNAsynthesis kit, the Uni-ZAP XR vector and the Gigapack II Gold packagingextracts were obtained from Stratagene Cloning Systems (USA). TheSequenase Version 2.0 kit for determining the nucleotide sequence waspurchased from the United States Biochemical Corporation (USA).

The resurrection plant Selaginella lepidophylla (Hook. & Grev. Spring.)was collected in dehydrated form from the rocky soil of the arid zonesof the States of Morelos and Oaxaca in Mexico. It was subsequentlycultivated in controlled conditions (24° C. and 16 hours of light withan average of 50% humidity) in Conviron growth chambers or in agreenhouse. The plants were watered every other day with 20 ml of waterfor 2 L flower pots. In order to treat S. lepidophylla to dehydrationstress, the complete plant or microphyll fronds were air-dried byplacing them on Whatman 3 MM filter paper. From that moment dehydrationtime was determined.

Strains

The cDNA bank was plated in the E. coli strain XL1-Blue MRF′ and thestrain SOLR was used to excise the pBluescript from the lambda phage,following the instructions given in the “ZAP-cDNA Synthesis Kit”(Strategene Cloning Systems, Calif. USA; catalogue #200400, 200401 and2004029). The E. coli DH5 alpha strain was used to subclone and makeconstructs. The A. tumefaciens LBA4404 strain was used to transformtobacco and the E. coli HB101 strain, carrying plasmid pRK2013 (Bevan,M. (1984) Nucl. Acids Res. 22: 8711-8721) was used to transfer plasmidpIBT36 from E. coli to A. tumefasciens by means of triparentalconjugation as previously described (Bevan, M. (1984), supra).

DNA Manipulation

Recombinant DNA techniques such as bacterial transformation, isolationof DNA from plasmid and lambda bacteriophage were carried out accordingto standard procedures (Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989) Molecular cloning: A laboratory manual. Second Edition. ColdSpring Harbor Laboratory press, New York). The labelling of radioactivefragments was carried out by the “random-printing” technique witholigonucleotides (Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem.132: 6.130.

Constructs

The expression vector pBN35 is a derivative of pBin19 (Bevan, M. (1984),supra) that was constructed subcloning the 850 bp of the cauliflowervirus CaMV 35S promoter (Guilley, H., Dudley, K., Jonard, G., Richards,K., & Hirth, L. (1982) Cell 21: 285-294) between the HindIII and SalIsites of pBin19 and the 260 bp fragment constituting the polyadenylationsignal of the T-DNA nopaline synthetase gene (Bevan, M., Barnes, W. &Chilton, M. D. (1983) Nucl. Acids Res. 11: 369-385) in the SacI andEcoRI sites of the same vector (FIG. 4).

The plasmid pIBT36 (FIG. 5) was constructed by subcloning the sl-tps/pcDNA in the BamHI and KpnI sites of the expression vector pBN35.

Construction of the cDNA Bank of S. lepidophylla

In order to isolate the cDNA clones an expression bank was prepared withmRNA isolated from S. lepidophylla microphylls dehydrated for 2.5 hours,using the ZAP cDNA synthesis kit, the Uni-ZAP XR vector and the GigapackII gold packaging extracts. The “ZAP-cDNA Synthesis Kit” laboratorymanual provided by the manufacturer (Stratagene Cloning Systems, Calif.,USA; catalogue #200400, 200401 and 2004029) was followed step by step.PolyA⁺ RNA was extracted from microphylls of S. lepidophylla, dehydratedfor 2.5 hours, in accordance with a known method (Chomczyniski, P. &Sacchi, N. (1987) Anal. Biochem. 162: 156-159). The initial titre of thebank was 2×10⁶ plaques of bacteriophage/ml and after amplification1.5×10¹¹ plaques of bacteriophage/ml.

The plasmid pBluescript SK (−) was excised from the bacteriophage bymeans of the “zapping” technique in accordance with the laboratorymanual “ZAP-cDNA Synthesis Kit” (Stratagene Cloning Systems, Calif.,USA; catalogue #200400, 200401 and 2004029).

DNA Sequencing

Nested deletions of the insert were created with enzymes ExoIII andNuclease S1 from the selected clone (Henikoff, S. (1984) Gene 28:351-359), in order to subsequently determine its nucleotide sequenceusing the chain termination method with dideoxynucleotides (Sanger, F.,Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467). The DNA sequence was analyzed using the University ofWisconsin Genetics Computer Group (UWGCG) software package (Devereux, J.Haeberli, P. & Smithies, 0. (1984) Nucl. Acids Res. 12: 387-395).Hydrophobicity plots were obtained using a known program (Kyte, J. &Doolittle, R. (1982) J. Mol. Biol. 157: 105-132) and protein sequencealignments with the BESTFIT program included in the UWGCG package.

Hybridization of the Nucleic Acids

In order to screen the bank, the bacteriophage plaques were transferredto a Hybond N⁺ nylon membrane (Amersham Life Sciences) which was treatedin accordance with the conventional method for denaturing DNA (Sambrook,J. et al., supra). Second Edition. Cold Spring Harbor Laboratory Press,New York]. The filter was hybridized with oligonucleotides, labelledwith the ³²P isotope by means of polynucleotide kinase, using 6×SSC(1×SSC=0.15 M NaCl and 0.015 M sodium citrate) at 37° C. The filter waswashed three times, 10 minutes each washing at the same temperature andunder the following conditions: 6×SSC; 4×SSC; and 2×SSC.

Southern and Northern gel blot techniques were performed according tostandard protocols (Sambrook, J. et al. supra) with the followingmodifications. For the genomic Southern, the DNA was fractionated on an0.8% agarose gel in TBE buffer and transferred to a Hybond N⁺ nylonmembrane (Amersham Life Sciences). The filter was hybridized usingsl-tps/p cDNA labelled with ³²P isotope as probe, using 2×SSC(1×SSC=0.15M NaCl and 0.015 M sodium citrate) at 65° C. The filter waswashed three times, twenty minutes each washing at the same temperature,and under the following conditions: 2×SSC; 1×SSC; and 0.5×SSC. For theNorthern, 1.2% agarose gel was used in a MOPS-formaldehyde buffer and aHybond N⁺ nylon membrane was also used for the transfer. Hybridizationconditions were in 50% formamide and 2×SSC at 42° C. The threesuccessive washings of the filter were performed with 2×SSC, 2×SSC and1×SSC, respectively at 55° C.

Transformation of Tobacco

The transformation of tobacco (Nicotania tabacum var. SR1) was carriedout by means of the leaf disk method (Horsch, R. B., Fry, J. E.,Hoffmann, N. L., Eichholtz, D., Rogers, S. G., Fraley, R. T. (1985)Science 227: 1229-1231), using Agrobacterium tumefasciens LBA4404containing the plasmid pIBT36. The leaf disks were cultivated in Petridishes containing MS medium with vitamins (Murashige, T. & Skoog, F.(1962) Physiol. Plant. 15: 473-497), hormones (0.1 ppm NAA an 1 ppm BAP)and antibiotics (100 μg/ml kanamycin and 200 μg/ml carbenicillin) toregenerate shoots in 4 to 6 weeks. Shoots were transferred to Magentapots containing Ms medium with antibiotics (100 μg/ml kanamycin and 200μg/ml carbenicillin) and without hormones nor vitamins, in order toregenerate roots in 2 to 3 weeks later. The regenerated plants weretransferred to pots with soil and cultivated in growth chambers (at 24°C. with 16 hours of light) in order to obtain fertile plants within 4 to6 weeks.

Trehalose Determination

Trehalose was determined by the degradative method with trehalase(Araujo, P. S., Panek, A. C, Ferreira, R. & Panek, A. D. (1989) Anal.Biochem. 176: 432-436). In order to obtain soluble sugars, 500 mg offresh tissue or 50 mg of dry tissue (frozen in liquid nitrogen) wereground in 0.5 ml of 100 mM PBS buffer, pH 7.0, in a homogenizer formicrocentrifuge tubes. Four volumes of absolute ethanol were added andthe samples boiled for 10 minutes in tubes with screwcap in order toavoid evaporation. Subsequently, they were centrifuged inmicrocentrifuge tubes for 2 minutes at 13,000 rpm and the supernatantwas recovered. Samples were reextracted again with the same volume of80% ethanol and the pellet was vacuum dried. The samples wereresuspended in 0.250 ml 50 mM PBS, pH 6.5.

For the determination of trehalose, 4 μl (c.a. 15 mU) of trehalase wereadded (Sigma cat. no. T-8778) to 10 to 30 μl of the extract, and it wasincubated for 2 hours at 30° C. As a negative control, a tube with anextract but without trehalase was used nd as a positive control a tubewith pure trehalose (Sigma no. cat. T-3663). The volume was brought to0.5 ml with 50 mM PBS, pH 7.0 and 0.5 μl of glucose oxidase andperoxidase from Sigma kit, cat. no. 510-A, were added in order todetermine glucose. Incubation was for 40 min. at 37° C. and the opticaldensity at 425 nm was immediately determined. In order to calculate theglucose concentration, a standard glucose curve was used with valuesbetween 0 and 75 mM. The values of the tubes without trehalase weresubtracted from those treated with this enzyme in order to calculate theamount of trehalose, taking into account that 1 mol of glucose is ½ molof trehalose.

Determination of the Enzymatic Activity

To determine the activity of trehalose-6-phosphate synthase, a reportedmethod was followed (Londesborough, J. & Vuorio. 0 (1991) J. Gen.Microbiol. 137: 323-330) that essentially consists of a coupled assaymeasuring the molar extinction of NADH at 340 nm. The reaction wascarried out in a volume of 100 μl containing 40 mM HEPES/KOH pH 6.8buffer, 10 mM glucose-6-phosphate, 5 mM UDP-glucose 10 mM MGCl₂ and 1mg/ml of bovine serum albumin. The reaction was incubated for 10 min. at30° C. and was stopped by boiling for 2 min. After cooling the tube, 900μl containing 40 mM HEPES/KOH pH 6.8 buffer, 10 mM MgCl₂, 2.5 μg/mlphosphoenolpyruvate, 0.24 mM NADH, 3.5 units of pyruvate kinase and 5units of lactate dehydrogenase (Sigma Cat. No. P-0294) were added. Thedisappearance of NADH at 340 nm, incubating under the same conditions asthose mentioned above, was measured spectrophotometrically. In order todetermine the specific activity of trehalose-6-phosphose synthase,protein concentration was measured by means of the Bradford method(Bradford, m. m. (1976) Anal. Biochem. 72: 248-254).

Example 1 Selection of TPS Genes

A suitable TPS gene can be selected in various manners. There are twomain possibilities to isolate plant TPS genes. First of all, functionalcomplementation of Saccharomyces cerevisiae cells that are deleted forthe TPS1 gene is a straightforward approach. Deletion of this genecauses a pleiotropic phenotype in yeast (Van Aelst et al., 1993, Mol.Microbiol. 8, 927-943). One of the phenotypes is that such cells cannotgrow on glucose. Construction of cDNA libraries from interesting plantsin yeast expression plasmids can be used to transform a yeast strainthat is deleted for TPS1. Transformants can than be checked for therestoration of growth on glucose. On the other hand, the synthesis oftrehalose in the transformants can also be measured. If transformantsare found that restore the growth on glucose medium or that produceagain trehalose, the plasmid DNA can be isolated and the insertssequenced. Based on the sequence it can then be concluded whether a realTPS homologue or a suppressor has been isolated.

Secondly, a comparison of the amino acid sequences oftrehalose-6-phosphate synthase, deduced from the reported nucleotidesequences, can be made. The sequences used come from E. coli, EC-otsA[Kaasen, I., McDougall, J., Strom, A. R. (1994) Gene 145: 9-15];Schizosaccharomyces pombe, SP-TPS1 [Blazquez, M. A., Stucka, R.,Feldman, H & Gancedo, C. (1994) J. Bacteriol. 176: 3895-3902];Aspergillus niger, AN-TPS1 [Wolschek, M. F. & Kubicek, C. P. (1994)NCBI: Seq. ID 551471; unpublished]; Saccharomyces cerevisiae, SC-TPS1[McDougall, J., Kaasen, Y. & Strom, A. R. (1993) FEMS Microbiol. Let.107: 25-30]; Kluyveromyces lactis, KL-GGS1 [Luyten, K., de Koning W.,Tesseur, Y., Ruiz, M. C., Ramos, J., Cobbaert, P., Thevelein, J. M.,Hohmann, S. (1993) Eur. J. Biochem. 217: 701-713].

As an example for the isolation of a plant homologue the isolation ofthe cDNA of S1-TPS will be described here.

Dehydrated resurrection plants, Selaginella lepidophylla, were collectedfrom rocky soil in arid zones of the States of Morelos and Oaxaca inMexico. They were subsequently cultivated in 2 L flower pots at 24° C.with 16 hours of light and 50% average humidity in Conviron growthchambers. The plants were watered every other day with 20 ml of water.

In order to isolate the cDNA clones, an expression bank was preparedusing 5 μg of mRNA isolated from 50 g of S. lepidophylla microphyllsdehydrated for 2.5 hours. After synthesizing the cDNA, it was clonedusing 1 μg of Uni-ZAP XR vector. The bacteriophages were packaged invitro and were subsequently screened with a mixture of degeneratedoligonucleotides that code for consensus regions intrehalose-6-phosphate synthase of the reported sequences of E. coli andyeast. One of the isolated clones corresponds to a cDNA (sl-tps) with acomplete coding region.

Analysis of the deduced amino acid sequence resulted in 53% identity fortrehalose-6-phosphate synthase and 29% for trehalose-6-phosphatephosphatase, as compared with reported sequences oftrehalose-6-phosphate synthase of bacteria and various yeasts.

Homology of the protein encoded by sl-tps, called SL-TPS, totrehalose-6-phosphate synthase, maps at the N-terminal region of theformer and the homology of SL-TPS to trehalose-6-phosphate phosphatasecan be found throughout the whole sequence.

The isolation procedure as described for S. lepidophylla can be used forany other plant, preferably monocots and dicots. This method is ofgeneral application because the degenerate oligos that were used to fishSelaginella TPS were tested successfully in Arabidopsis thaliana. Usinga PCR reaction a fragment of the A. thaliana TPS gene could be isolated.Based on this fragment the complete A. thaliana TPS gene was isolated.

Example 2 Preparation of Constructs

1. Construction of Yeast Expression Vectors Containing Plant TPS Genes.

A 3.1-kb fragment containing the full length SlTPS1 gene was obtainedafter amplification by PCR (94° C., 3 min, 1 cycle; 94° C., 1 min, 50°C., 1 min, 72° C., 2 min, 40 cycles; 72° C., 10 min, 1 cycle) usingExpand High-fidelity DNA polymerase (Boehringer). As oligonucleotides,SLTPS-S1 (5′-CATGCCATGGCTATGCCTCAGCCTTACC-3′, (SEQ ID NO: 1) boldindicates initiation codon and underlined NcoI site) and universal(5′-GTAAACGACGGCCAGT-3′) (SEQ ID NO: 2) primers were used with S1-TPS1cDNA cloned in pBluescript SK as template. The PCR-fragment was digestedwith NcoI and KpnI and cloned in pSAL4. Yeast tps1) and tps1) tps2)mutant strains were transformed and selected on SDGal (-ura) plates.Complementation was assayed in SDG1c (-ura minimal medium plus 100 μMCuSO₄).

For the construction of the N-terminal deletion construct the followingoligonucleotides were used:

oligo 5′ SLTPS-100 5′-CATGCCATGGGTCGAGGCCAGCGGTTGC-3′ (SEQ ID NO: 3),bold indicates initiation codon and underlined NcoI site.

oligo 3′ universal 5′-GTAAACGACGGCCAGT-3′ (SEQ ID NO: 2).

A 2.8-kb fragment was obtained after amplification by PCR (94° C., 3min, 1 cycle; 94° C., 1 min, 50° C., 1 min, 72° C., 2 min, 40 cycles;72° C., 10 min, 1 cycle) using Expand High-fidelity DNA polymerase(Boehringer) with oligos SLTPS-100 and universal, and S1-TPS1 cDNAcloned in pBluescript SK as template. The PCR-fragment was digested withNcoI and KpnI and cloned in pSAL4. Yeast tps1Δ and tps1Δ tps2Δ mutantstrains were transformed and selected in SDGal (-ura). Complementationwas assayed in SDGlc (-ura minimal medium plus 100 μM CuSO₄).

For the construction of yeast expression vectors containing the A.thaliana TPS gene RT-PCR was used.

Total RNA (5 μg) extracted from Arabidopsis thaliana cv. Columbiaseedlings grown for 2 weeks in liquid MS medium containing 100 mM NaCl,was reversed transcribed using SuperScript II (GIBCO) using an oligo dT(25 mer) primer. A PCR reaction (94° C., 3 min, 1 cycle; 94° C., 1 min,50° C., 1 min, 72° C., 2 min, 40 cycles; 72° C., 10 min, 1 cycle) wasdone using Expand High-fidelity DNA polymerase (Boehringer) to amplifyAtTPS1 using oligos Ath/TPS-5′(5′-CATGCCATGGCTATGCCTG-GAAATAAGTACAACTGC-3′ (SEQ ID NO: 4); boldindicates initiation codon, underlined is NcoI site) and Ath/TPS-3′(5′-ATAGTTTTGCGGCCGCTTAAGGTGAG-GAAGTGGTGTCAG-3′ (SEQ ID NO: 5); boldindicates termination codon, underlined NotI site). A 2.8-kb fragmentwas obtained corresponding to the expected size, digested with NcoI andNotI, and cloned in pSAL6. Yeast tps 1) and tps 1) tps2) mutant strainswere transformed. Transformants were grown in SDGal (-his minimalmedium). Complementation was assayed in SDG1c (-his minimal medium plus100 μM CuSO₄).

For the construction of the N-terminal deletion construct the followingoligonucleotides were used:

oligo Ath/TPS-)N5′

5′-CATGCCATGGCTTATAATAGGCAACGACTACTTGTAGTG-3′ (SEQ ID NO: 6), boldindicates initiation codon and underlined NcoI site.

oligo Ath/TPS-3′

5′-ATAGTTTTGCGGCCGCTTAAGGTGAGGAAGTGGTGTCAG-3′ (SEQ ID NO: 5); boldindicates termination codon, underlined NotI site.

Total RNA (5 μg) extracted from Arabidopsis thaliana cv. Columbiaseedlings grown for 2 weeks in liquid MS medium containing 100 mM NaCl,was reverse transcribed using Superscript II (GIBCO) and an oligo dT (25mer) primer. A PCR reaction (94° C., 3 min, 1 cycle; 94° C., 1 min, 50°C., 1 min, 72° C., 2 min, 40 cycles; 72° C., 10 min, 1 cycle) was doneusing Expand High-fidelity DNA polymerase (Boehringer) to amplify AtTPS1using oligos Ath/TPS-?N5′ and Ath/TPS-3′. A 2.6-kb fragment was obtainedcorresponding to the expected size, digested with NcoI and NotI, andcloned in pSAL6. Yeast tps1Δ and tps1Δtps2Δ mutant strains weretransformed. Transformants were grown in SDGal (-his minimal medium).

Complementation was assayed in SDGlu (-his minimal medium).

2. Construction of Plant Expression Vectors Containing Plant TPS Genes

To clone in plant expression vectors, plant trehalose-6-phosphatesynthase genes were first tested in a yeast tps1Δ mutant andsubsequently subcloned in appropriate plant transformation vectors. The2.9-kb AtTPS1 and 2.6-kb ΔNAtTPS1 coding regions were isolated afterdigestion of plasmids pSAL6::AtTPS1 and pSAL6::ΔNAtTPS1 with NcoI andKpnI enzymes. This latter site is downstream of NotI site in pSAL6.

The 3.1-kb S1TPS1 and 2.8-kb)NS1TPS1 coding regions were isolated afterdigestion of plasmids pSAL4.S1TPS1 and pSAL4.dNS1TPS1 with NcoI and KpnIenzymes, as well. All DNA fragments were ligated to a 57-bp fragmentcontaining AtTPS1 5′ leader, XbaI and NcoI sites. This fragment wasobtained after annealing oligonucleotides NA4(5′-CTAGAGCGGCCGCCAGTGTGAGTAATTTAGTTTTGGTTCGTTTTGGTGT GAGCGTC-3′) (SEQID NO: 7) and NA5(5′-CATGGACGCTCACACCAAAACAGAACCAAA-ACTAAATTATCACACTGGCGGCCGCT-3′) (SEQID NO: 8).

Each ligated leader-coding-region cassette was further ligated to theexpression vector pBN35 digested with XbaI and KpnI (FIG. 1) leading toplasmids pIBT101 containing AtTPS1 (FIG. 2), pIBT102 containing ΔNAtTPS1(FIG. 3), pIBT103 containing S1TPS1 (FIG. 4) and pIBT104 containingΔNS1TPS1 (FIG. 5). The vector pBN35 allows the expression of any geneunder the control of the cauliflower virus (CaMV) 35S promoter (Guilley,H., et al., Cell 21: 285-294 (1982)) which is a strong and constitutivepromoter.

These plasmids were used to obtain transgenic plants, transformed bymeans of the Agrobacterium system, that when regenerated were capable ofproducing trehalose. These constructs can be expressed in any plant thatcan be transformed using the Agrobacterium system or by any other methodknown in the state of the art.

The expression vector pBN35 is a derivative of pBin19 (Bevan, M., Nucl.Acids Res. 22: 8711-8721 (1984)) that was constructed by subcloning the850 bp of the cauliflower virus CaMV 35S promoter (Guilley, H. et al.,supra) between the HindIII and SalI sites of pBin19 and the 260 bpfragment constituting the polyadenylation signal of the T-DNA nopalinesynthetase gene (Bevan, M. et al., Nucl. Acids Res. 11: 369-385 (1983))in the SacI and EcoRI sites of the same vector (FIG. 1).

3. Construction of HA-Tagged S1 TPS1, At TPS1, ΔN S1 TPS1 and ΔN At TPS1Alleles.

In order to determine whether the difference in activity between thefull length plant TPS1 genes and the N-terminally deleted alleles iscaused by the fact that the former ones are not able to form a correctTPS complex when expressed in yeast cells, we have made tagged versionsof these genes. The scheme as shown in FIG. 9 was used to make theseconstructs. FIG. 10 shows the resulting plasmids.

The plasmid containing the plant TPS genes is digested with two uniquerestriction sites, one cutting just after the gene (R1) and the secondcutting close to the 3′ of the gene (R2). The fragment that is removedby the combined use of R1 and R2 is replaced with a fragment obtained byPCR that is also digested with R1 and R2. The R2 site is located withinthe PCR product whereas the R1 site is part of the reverse primer (rev).The constitution of the reverse primer is as follows:

5′ R1-STOP-HAtag-Codons for 6 amino acids of relevant gene-3′

Table 1 shows the primers that we have used:

The primers can be used for both the full length genes as well as the ΔNalleles.

TABLE 1 5′ primers 3′ primers S1 HA 5′: S1 HA 3′:5′CGACTACGTCCTTTGCATAGG 5′ CT GGT ACC TCA TGC GTA ACAC3′GTC AGC GAC ATC ATA CGG (SEQ ID NO: 9) ATA CTG TAC CGC TGG AGC GAG 3′(SEQ ID NO: 10) At HA 5′: At HA 3′: 5′GACGTCCTTCACCAGAGAAGA 5′CT GCA TGC TCA TGC GTA TCTC3′ GTC AGG CAC ATC ATA CGG (SEQ ID NO: 11)ATA AGG TGA GGA AGT GGT GTC 3′ (SEQ ID NO: 12)In table 2 the different vectors and restriction enzymes that were usedare indicated.

TABLE 2 PCR product Restriction enzyme: (template): Vector: R1 R2 Sl(pSal4 vector) pSal4/Sl TPS1 KpnI PflmI pSal4/ΔN SI TPS1 At (pSAL6vector) pSal6/At TPSl SphI AspI pSa16/ΔN At TPS1

Example 3 Functional Complementation of Yeast Tps Mutants with ModifiedPlant TPS Genes

1. Complementation of the Growth Defect of tps1Δ and tps1Δtps2Δ Strains

Tps1Δ and tps1Δtps2Δ strains were transformed with yeast expressionvectors containing either full length or N-terminal deleted S.lepidophylla or A. thaliana TPS genes. As controls, Wild Type, tps1Δ ortps1Δtps2Δ strains were transformed with an empty plasmid, or with aplasmid containing the yeast TPS1 gene. Transformants were tested forgrowth on glucose and fructose containing medium. FIG. 6 shows thegrowth on glucose and fructose containing medium of Wild Type and tps1Δstrains transformed with a yeast expression vector containing a completeor the truncated S. lepidophylla TPS gene. The lanes are as follows: 1.WT, 2. tps1Δ, 3. tps1Δ+ANTPS1 S1, 4. tps1Δ+TPS1 S1, 5. tps1Δ+TPS1S, 6.tPS1ΔtPs2Δ, 7. tPS1ΔtPs2Δ+ΔNTPS1 S1, 8. tPs1ΔtPS2Δ+TPS1 S1, 9.tps1Δtps2Δ+TPS1 Sc, 10. tps1Δtps2Δ+TPS2 Sc.

The full length clone can only complement the tps1Δtps2Δ strain. Itcannot complement the growth defect of a tps1Δ strain on glucose orfructose. However, if the N-terminal part is deleted, the S.lepidophylla gene expressed from the Cu-inducible promoter cancomplement the growth defect in a tps1Δ strain. This clearly illustratesthe beneficial effect of the N-terminal deletion.

If the plant gene is expressed under the control of a stronger promoter,also the full length clone can complement the tps1Δ strain for growth onglucose or fructose (not shown).

2. Restoration of Trehalose Levels in tps1Δ Strains

Trehalose was measured in yeast cells using the method of Neves et al.(Microbiol. Biotechnol. 10: 17-19 (1994)). In this method, trehalose isdegraded by trehalase and the glucose that is formed is measured by theglucose oxidase/peroxidase method. Briefly, cells were collected on afilter, with pores of 0.22 or 0.45 μm, on a vacuum flask and washed withwater. Cells were collected, the weight was determined and they werefrozen in liquid nitrogen. For extraction of the trehalose; Na₂C0₃(0.25M) was added to the cells (1 ml per 50 mg of cells) and they wereboiled for 20 min. After centrifugation, 10 μl of the supernatant wasused to measure the trehalose content. Each sample was neutralized byadding 5 μl of a 1M acetic acid solution. To each sample 5 μl of bufferT1 (300 mM NaAc+30 mM CaCl₂ pH 5.5) and 20 μl of trehalase solution(isolated from the fungus Humicola grisea) were added. The samples wereincubated at 40° C. for 45 min. In this step, trehalose is broken downto glucose. In parallel to the samples, trehalose standards and controlsamples were also measured. After this incubation the tubes werecentrifuged briefly and 30 μl of the supernatant was used for glucosedetermination.

To each sample, 1 ml of glucose oxidase/peroxidase solution containingo-dianisidine (0.1 g/ml) was added and the mixture was incubated for 1 hat 30° C. The reaction was stopped by the addition of 56% (v/v) sulfuricacid. For each sample the extinction at 546 nm was measured.

The trehalose levels were measured in S. cerevisiae tps1Δ strainstransformed with plasmids containing the S1TPS, ΔNS1TPS1, AtTPS1 orΔNAtTPS1 genes under the control of a Cu-inducible promoter. Table 3shows the results of three independent experiments. The abbreviation 1Δstands for tps1Δ.

TABLE 3 trehalose TPS activity μmol/g ww μkat/g protein WT + pSAL4galactose 58.4 0.63 glucose 48.6 0.51 fructose 72.5 0.58 1 Δ + pSAL4galactose 3.2 0.05 glucose NG NG fructose NG NG 1 Δ + pSAL4::SITAS1galactose 2.1 0.01 glucose NG MG fructose NG NG 1 Δ + pSAL4::ΔN SITPS1galactose 43.5 0.41 glucose 49.2 0.65 fructose 89.2 0.43 1 Δ +pSAL6::AfTPS1 galactose 1.6 0 glucose NT NT fructose NT NT 1 Δ +pSAL6::ΔN AtTPS1 galactose 36.5 0.1 glucose 41.4 0.12 fructose 59 0.07 1Δ + pSAL4::ScTPS1 galactose 43.4 1.25 glucose 37.1 1.01 fructose 54.41.53 1 Δ + pSAL4::ΔC SlTPS1 galactose 0 0 glucose KG NG fructose NG NG 1Δ + pSAL4::ΔNΔC Sl TPS1 galactose 8.2 0.08 glucose 48.2 NT fructose NTNTThe results in this table confirm the results shown in FIG. 6. The fulllength clones cannot complement the growth defect of a tps1Δ strain onglucose or fructose containing medium. Furthermore on galactose thesefull length clones are not capable of producing trehalose in tps1Δstrains. However, if the N-terminal part of either the S. lepidophyllaor the A. thaliana TPS gene is deleted and the tps1Δ strains aretransformed with plasmids containing these genes, these strains are ableto grow on glucose or fructose and these strains also produce highlevels of trehalose. (“−” means that no measurement could be madebecause the cells are unable to grow under this condition; “ND” meansnot detectable)3. Deletion of the N-Terminus is Necessary to Obtain TPS Activity in anIn Vitro Assay.

Trehalose-6-phosphate synthase activity was measured by a coupled enzymeassay as described by Hottiger et al. (J. Bacteriol., 169: 5518-5522(1987)). Crude extracts were desalted on a Sephadex G-25 column with abed volume of 2 ml, pre-equilibrated with 50 mM Tricine buffer pH 7.0.The assay mixture contained 50 mM Tricine/KCl (pH 7.0), 12.5 mM MgCl₂, 5mM UDP-glucose, 10 mM glucose-6-phosphate, enzyme sample and water in atotal volume of 240 μl. In controls, glucose-6-phosphate was omitted andreplaced by water. The assay mixtures were incubated at 30° C. for 30min. The reaction was stopped by boiling for 5 min. After cooling, assaymixtures were centrifuged at 13000 rpm for 10 min. The UDP formed in thesupernatant was measured enzymatically. The assay mixture contained 66mM Tricine/KCl (pH 7.6), 1.86M phosphoenolpyruvate, 0.3 mM NADH, 5Ulactic dehydrogenase, and 60 μl of sample in a total volume of 990 μl.The reaction was started by addition of 10 μl pyruvate kinase, andincubated at 37° C. for 30 min. The decrease in absorbance at 340 nm wasrecorded and used to calculate the enzyme activity.

$\frac{{{enzyme}\mspace{14mu}{{activity}( {\mu\;{kat}\text{/}{{gprot}.}} )}} = {\Delta\;{OD}_{340} \times {240\hat{}1} \times 10^{12}}}{\begin{matrix}{{60\mspace{14mu}\mu\; 1 \times 6},{22 \times 10^{6} \times 30\mspace{14mu}\min \times}} \\{{60\mspace{14mu} s\text{/}\min \times 30\mspace{14mu}\mu\; l \times {mg}\text{/}{ml}\mspace{14mu}{protein}\mspace{14mu}{lkat}} = {6 \times 10^{7}\mspace{14mu}{{units}.}}}\end{matrix}}$

The results of the TPS activity measurements are shown in Table 3. Theyindicate that only the N-terminal deleted TPS genes, and not the fulllength clones, result in high activity of trehalose-6-phosphate synthasewhen expressed in yeast.

After construction of the HA-tagged alleles these alleles wereintroduced in tps1Δ and tps1Δtps2Δ strains. The following strains wereobtained:

PVD164: a leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1

can1-100 GAL SUC2+tps1Δ::TRP1+pSAL4/S1 TPS1 HAtag (URA3)

PVD165: a leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1

can1-100 GAL SUC2+tps1Δ::TRP1+pSAL4/?N Si TPS1 HAtag

(URA3)

PVD179: a leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1

can1-100 GAL SUC2+tps1Δ::TRP1

tps2Δ::LEU2+pSAL4/S1 TPS1 HAtag (URA3)

PVD181: a leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1

can1-100 GAL SUC2+tps1Δ::TRP1

tps2Δ::LEU2+pSAL4/ΔN S1 TPS1 HAtag (URA3)

The presence of the HA-tag did not interfere with the function of theplant genes. Expression from the CUP1 promoter (pSal vectors) of thefull length plant alleles did not restore the growth defect on glucoseof a tps1Δ strain. Expression of the N-terminally deleted alleles didrestore growth on glucose as we have seen for the non-HA tagged alleles.

The expression of the full length and N-terminally deleted HA-tagged S1TPS1 genes was tested in both the tps1Δ and the tps1Δtps2Δ strain.

Strains PVD164 (tps1Δ+S1 TPS1-HA), PVD165 (tps1Δ+ΔN S1 TPS1 HA), PVD179(tps1Δ tps2Δ+S1 TPS1-HA) and PVD181 (tps1Δ tps2Δ+ΔN S1 TPS1-HA) havebeen grown till stationary phase in SDgal-URA (+CuSO₄). The cells werewashed in extraction buffer (for 1 liter: 10.7 g Na₂HP0₄.2H₂O, 5.5 gNaH₂PO₄.H₂O, 0.75 g KCl, 246 mg MgSO₄.7H₂O, 1 mM PMSF, pH 7.0) andresuspended in 500 μl extraction buffer. Extracts were made by vortexing2 times for 1 min in the presence of glass beads. The extracts werecleared by centrifugation for 20 min.

10 μg of the extracts were run on a 7.5% PAGE gel. After blotting, thenitrocellulose membranes were incubated for 1 h in TBST containing 2%BSA (5×TBS: 6 g Tris+45 g NaCl, pH 7.4; TBST=1×TBS+0.05% Tween20). Thefilters were then incubated for 1 h with anti-HA antibodies (anti-HAhigh affinity, rat monoclonal antibody clone 3F10, Boehringer Mannheim)diluted 1:1000 in TBST containing 2% BSA. The filters were washed 3×5min in TBST and subsequently incubated for 45 min with the secondaryantibody (Sigma A-6066; anti-rat) diluted 1:20000 in TBST containing 2%BSA. The filters were then washed 3×5 min in TBST. Afterwards, thefilters were washed for 5 min in TBS. The alkaline phosphatasedeveloping mixture (10 ml 100 mM Tris, pH 9.5; 50 mM MgCl₂; 100 mM NaCl,37.5 μl X-phosphate and 50 μl nitro blue tetrazolium (NBT)) was added tothe filters and when the bands became visible the reaction was stoppedby adding H₂O. The results are presented in FIG. 11.

The calculated molecular weight of the full length S1 Tps1 protein(without the HA tag) is 109353 whereas the ΔN S1 Tps1 protein has amolecular weight of 99453.

In order to find out whether the difference in complementation capacitybetween the full length TPS1 gene and the N-terminally deleted TPS1 geneis caused by the fact that the full length protein can not make acorrect TPS complex FPLC analysis of yeast extracts prepared from tps1Δstrains transformed with either the full length S1 TPS1 or the ΔN S1TPS1 gene was performed. The extracts were separated on a gelfiltrationcolumn (Superdex 200 HR 10/30) and fractions of 750 μl were collected asdescribed by Bell et al., (J. Biol. Chem., 373, 33311-33319, 1998). Thefirst fraction that contains proteins is fraction 10. Based on thecolumn characteristics and based on calibration experiments the proteinsin fraction 10-14 correspond to very large protein complexes rangingfrom 800 000 to 400 000 daltons.

FIG. 11 gives the result of the western blot using anti-HA antibodies.Here we only show fractions 10 to 15. The free TPS1 protein is presentin fractions 25-27 (not shown). Very important is the fact that both thefull length and the ΔN S1 TPS1 alleles are able to form complexes withthe other subunits of the TPS complex. This might indicate that theN-terminal region itself may exert an inhibitory function directly onthe remainder of the plant Tps1 protein.

The fact that full length TPS1 alleles do not result in any trehalosesynthesis in higher plants may be caused by the inhibitory effect of theN-terminus. Construction of transgenic plants with these N-terminallydeleted constructs may result in plants with higher trehalose levels andbetter stress resistance.

Example 4 Construction of Transgenic Plants of Arabidopsis thaliana,Producing Trehalose

The transformation of Arabidopsis thaliana ecotype Columbia is carriedout by means of the vacuum infiltration method (Bechtold, N. et al., C.R. Acad. Sci. Paris 316: 1194-1199 (1993); Bent, A. et al., Science 265:1856-1860 (1994)), using Agrobacterium tumefasciens C58C1 strainharboring the helper plasmid pMP90 and the appropriate plasmidconstruct, and is mobilized from E. coli strain DH5-alpha by triparentalmating using E. coli strain HB101 harboring plasmid pRK2013 as a helper.

Briefly, A. thaliana plants are grown at 22-20° C., under 16 hrs light,for 4-6 weeks, until inflorescences are starting to appear. Afterpouring an Agrobacterium culture inside a vacuum desiccator, potscontaining Arabidopsis plants are placed upside down and vacuuminfiltrated for 5 min. Plants are allowed to grow under the conditionsdescribed above. Seeds are harvested and selected in Petri dishescontaining 4.4 g/L MS salts, 1% sucrose, 0.5 g/L MES buffer pH 5.7(KOH), 0.8% phytagar, and 30 mg/L kanamycin to select transformants. 500mg/L carbenicillin are added as well to stop bacterial growth. After 5-7days transformants are visible as green plants and transferred to thesame medium as above but with 1.5% phytagar. After 6-10 days plants withtrue leaves are transferred to soil.

Analysis of transgenic plants is conducted to determine gene integrationin the plant genome and gene copy-number by Southern blot andtranscription of transgene is carried out by Northern blot by standardtechniques (Sambrook, J. et al., Molecular cloning: A laboratory manual.Second Edition. Cold Spring Harbor Laboratory Press, New York (1989)).An antibody against S1-TPS1 is used to confirm the correct translationof transgene using Western blot (Towbin, H. et al., Proc. Natl. Acad.Sci. USA 76, 4350-4353 (1979)). Trehalose-6-phosphate synthase activity(De Virgilio, C. et al., FEBS Lett. 273: 107-110 (1990)) and trehalosecontent (Neves, M J. et al., World J. Microbiol. Biotechnol. 10: 17-19(1994)) were measured by known methods.

Table 4 shows the phentotypes of transgenic Arabidopsis thalianaoverexpressing plant trehalose-6-P synthase gene.

TABLE 4 PHENOTYPES OF TRANSGENIC Arabidopsis thaliana OVEREXPRESSINGPLANT TREHALOSE-6-P SYNTHASE GENES. GROWTH (FLOWERING INFLO- TRANSGENEROSETTE TIME) RESCENCES SEEDS SILIQUA AtTPSl Larger with Delayed LargerNormal Normal green and (1-2 weeks) some purple leaves ΔNAtTPS1 Smallerwith Delayed Larger Fewer Normal on many purple (2-3 weeks) andinflorescence leaves purple tips and smaller and sterile oninflorescence bottoms S1TPS1 Smaller with Delayed Larger Normal Normalgreen and (1-2 weeks) some purple leaves ΔNS1TPS1 Smaller with DelayedLarger Fewer Normal on many purple (2-3 weeks) and inflorescence leavespurple tips and smaller and sterile on inflorescence bottoms CONTROLNormal Normal Normal Normal Normal

Example 5 Mass Synthesis of Trehalose in Transgenic Potato, Sugarbeetand Sugarcane Plants

Genetic engineering has made it possible to express almost any gene in aheterologous organism.

Transgenic plants can be used as bioreactors for the large-scaleproduction of compounds of commercial interest, that are normally onlyobtained in limited amounts, such as biodegradable plastics, differentcarbohydrates, polypeptides for pharmaceutical use and enzymes forindustrial use (Goddijn, O. J. M. and Pen, J., Trends Biotech. 13:379-387 (1995)). Different methods have been reported for thetransformation of higher plants, including crops of great economicimportance (Walden, R. and Wengender, R., Trends Biotech. 13: 324-331(1995)). The transformation of tobacco (Horsch, R. B. et al., Science227: 1229-1231 (1995)) and potato (Sheerman, S, and Bevan, M. W., PlantCell Rep. 7: 13-16 (1988)) are efficiently carried out using theAgrobacterium tumefasciens system and said technique can be set up in alaboratory by persons who master the state of the art. The constructsfor the expression in plants of any plant trehalose-6-phosphate_synthasegene, preferably S1TPS1, devoid of its N-terminal region can be made ina vector derived from the Ti plasmid that lacks the T-DNA tumorigenicgenes and which contains a selection marker for transformed plants that,for example, confers resistance to kanamycin (Bevan, M., 1984, Nucl.Acids Res. 22: 8711-8721). Furthermore, an appropriate promoter must bechosen depending on the use that will be given to the transgenic plants.The polyadenylation signal from the nopaline synthetase gene in theT-DNA can be used (Bevan, M., Barnes, W. and Chilton, M.-D., 1983, Nucl.Acids. Res. 11: 369-385).

To overproduce trehalose for industrial use, plants such as potato,sugarcane or sugarbeet, can be used. For instance, potato stores largeamounts of carbohydrates in the tuber. In terms of the plant biomass,potato represents one of the most productive crops per unit of area(Johnson, V. A. and Lay, C. L., 1974, Agric. Food Chem. 22: 558-566).There are strong tuber-specific promoters such as the class-1 promoterof patatin gene (Bevan, M. et al., Nucl. Acids Res. 14: 4625-4638(1986); Jefferson, R. et al., Plant Mol. Biol. 14: 995-1006 (1990)) thatcould be used to produce large amounts of trehalose. The convenience ofusing potato, sugarbeet or sugarcane as systems for overproducingtrehalose is that these plants are human food and therefore trehaloseisolated from them would be easily accepted by consumers. The trehaloseobtained by overexpression in plants could be used to preservebiomolecules for industrial use, such as restriction and modificationenzymes (Colaco, C. et al., Bio/Technology 10: 1007-1111 (1992)),vaccines, or processed foods.

Example 6 Transgenic Cereal Plants Resistant to Environmental Stress

The cereals constitute the basic food for world nutrition and they couldbe cultivated under unfavourable weather conditions if they couldproduce trehalose in response to cold, heat, salinity or drought. Inorder to achieve this, it is required to express any planttrehalose-6-phosphate_synthase gene, preferably S1-TPS1, devoid of itsN-terminal region under the control of promoters that are induced by anyof these environmental factors (Baker, S. S. et al., Plant Mol. Biol.24: 701-713 (1994); Takahashi, T. et al., Plant J. 2: 751-761 (1992);Yamaguchi-Shinozaki, K. and Shinozaki, K., Plant Cell 6: 251-264(1994)). The synthesis of trehalose only under stress conditions wouldavoid the continuous production of trehalose (using a constitutivepromoter) which diverts the carbohydrate metabolism and as a consequencecould decrease the quality and productivity of grains. There are reportson transformation of maize (D'Halluin, K. et al., Plant Cell 4:1495-1505 (1992)), barley (Wan, Y. and Lemaux, P. G., Plant Physiol.104: 37-48 (1994)), wheat (Vasil, V. et al., Bio/Technology 10: 667-674(1992)) and rice (Shimamoto, K. et al., Nature 338: 274-276 (1989)).This methodology can be implemented by a person familiar with the stateof the art.

Example 7 Fruit from Transgenic Plants with a Longer Shelf Life

Different types of fruit such as tomato, mango and banana ripen quicklyand tend to rot before they reach consumers. The early harvesting offruits and their storage in refrigeration or in chambers with acontrolled environment has been traditionally used to avoid thisproblem. However, these methods are expensive, especially if the fruitswill be transported to distant places. In order to increase the shelflife of tomato, a delay in ripening has been reported using transgenicplants that express in antisense the polygalacturonase gene which isinvolved in fruit ripening. In spite of this delay in ripening, there isstill the problem that after a certain time this process is carried outwithout the product necessarily reaching the consumer in a good state.

As an alternative to this method, it is here proposed to producetrehalose in transgenic tomato, mango and banana plants. For example,using a specific promoter of the tomato fruit (Bird, C. R. et al., PlantMol. Biol. 11: 651-662 (1988)), any plant trehalose-6-phosphate synthasegene, preferably S1-TPS1, devoid of its N-terminal region could beoverexpressed so that trehalose accumulates specifically in that organ.The method of transformation and regeneration for tomato plants (asdescribed by McCormick, S. et al., Plant Cell Rep. 5: 81-84 (1986)) canbe carried out by any person who knows that state of art. Tomato andother types of fruit could be harvested ripe and then submitted whole orin parts to desiccation and preserved for long periods without the needfor refrigeration. When rehydrated, the fruit would have the normalorganoleptic properties that the consumer demands. In principle, thestrategy described above can be implemented for other types of fruitprovided a regeneration and transformation system is available for theplant in question and that there is an appropriate fruit-specificpromoter.

Example 8 Increase in the Viability of Cells, Organs or Plant PartsInvolved in Sexual or Asexual Reproduction

The production of pollen with prolonged viability would be of great helpin plant breeding programs and in the preservation of germplasm.Similarly, the possibility of storage for long periods and an increasein the viability of seeds, bulbs, tubers, cuttings for grafts, sticksand flowers will have a great impact on plant breeding and theconservation of germplasm. The presence of trehalose in a tissue, organor part of the transformed plant will make it possible to preserve saidtissue, organ or part at room temperature in a dehydrated state forsignificantly greater periods than without trehalose. In order toachieve this objective, it is necessary to clone any planttrehalose-6-phosphate_synthase gene, preferably S1-TPS1, devoid of itsN-terminal region into a plant expression vector under a tissue-specificor organ-specific promoter and transform the plant in question with thisconstruct by any of the reported methods known to someone familiar withthe state of art. There are reports of pollen-specific promoters(Guerrero, F. D. et al., Mol. Gen. Genet. 224: 161-168 (1990),tuber-specific promoters (Bevan, M. et al., Nucl. Acids Res. 14:4625-4638 (1986); Jefferson, R., Plant Mol. Biol. 14: 995-1006 (1990))and seed-specific promoters (Colot, V. et al., EMBO J. 7: 297-302(1988)) that could be used to construct hybrid genes for the expressionof trehalose in plants.

Example 9 Testing of Different Stress Conditions

The tolerance of the transgenic plants against various stress conditionswas tested in the following manner.

1. Cold

Transgenic plants overexpressing S1-TPS1 under control of theconstitutive 35S promoter were analyzed by Southern blot to verifycorrect transgene insertion in the plant genome. S1-TPS1 gene expressionwas corroborated by Northern blot. Transgenics were checked toaccumulate trehalose and not detected in control plants, transformedonly with the vector. Transgenic Arabidopsis T2 generation plants (20days old) were grown in pots containing soil/vermiculite, under 16 hrlight/8 hr dark at 24° C. in a growth chamber under well-wateredconditions. Control and trehalose-synthesizing plants were transferredto a growth chamber at 4° C. for 10 days at constant light and returnedto 24° C. for 2 days. Other sets of plants were left at 24° C. Damage incold-treated plants was estimated visually (chlorosis, leafdeterioration and death) and by measuring photoinhibition ofphotosynthesis (Murata, N. et al., Nature 356: 710-713 (1992)) using anIRGA (infrared gas analyzer) apparatus. Also growth retardation wasmeasured in leaf size ad plant height after comparison of cold-treatedplants vs. non-treated.

2. Freezing

Freezing tolerance was determined in cold-treated control andtrehalose-synthesizing plants, obtained as previously described, by theelectrolyte leakage test. Detached leaves were frozen to various subzerotemperatures and, after thawing, cellular damage due to membrane lesionswas estimated by measuring ion leakage from the tissues using aconductivity meter (Jaglo-Ottosen, K. R. et al., Science 280:104-106(1998).

3. Heat

Transgenic Arabidopsis T2 generation plants (20 days old), control andtrehalose-synthesizing were grown in pots containing soil/vermiculite,under 16 hr light/8 hr dark at 24° C. in a growth chamber underwell-watered conditions. Plants were preacconditioned after incubationfor two hr at 35° C. and then subjected to 1 hr of heat stress atvarious temperatures ranking from 46 to 56° C. for each independenttreatment. Plants were returned to 24° C. for 5 days. Damage wasdetermined visually (chlorosis, leaf deterioration and death). Similartests can be conducted using plantlets grown for 7 days at 24° C. onmoisted filter paper inside Petri dishes (Lee, J. H. et al., Plant J. 8:603-612 (1995)).

4. Dessication

Transgenic Arabidopsis T2 generation plants (20 days old), control andtrehalose-synthesizing were grown in pots containing soil/vermiculite,under 16 hr light/8 hr dark at 24° C. in a growth chamber underwell-watered conditions. Drought stress was imposed by stopping thewatering for several days until the leaves were wilted and then theplants were rewatered. Controls did not recover whereastrehalose-producing plants continued growing normally. Also detachedleaves were air-dried at 20% relative humidity. The fresh weight wasmeasured over a 48-hr period. Trehalose-producing plants lost lessweight (Holmstrom, K.-O. et al., Nature 379: 683-684 (1996)).

5. Osmotic Stress

Transgenic Arabidopsis T2 generation plants (20 days old), control andtrehalose-synthesizing were grown in pots containing soil/vermiculite,under 16 hr light/8 hr dark at 24° C. in a growth chamber underwell-watered conditions. Independent sets of plants were irrigated withvarious concentrations of NaCl (100-300 mM) or PEG (5 or 10%) during 1-3weeks. Plant growth was evaluated by measuring the percent of change inheight and in fresh weight (Tarczynski, M. C. et al., Science259:508-510 (1993)).

6. Storage

The plant TPS gene will be cloned under the control of for instance thetomato fruit-specific E8 promoter which is induced by ethylene and itsmaximum activity is reached in mature fruits (Lincoln, J. E. & Fischer,R. L., Mol. Gen. Genet. 212: 71-75 (1988); Good, X. et al., Plant Mol.Biol. 26:781-790 (1994); Tieman, D. et al., Plant Cell 4: 667-679(1992)) in pBIN19 vector containing a NOSpA 3′ end (Guilley, H., et al.,Cell 21: 285-294 (1982)) and (Bevan, M. et al., Nucl. Acids Res. 11:369-385 (1983)). The vectors will be mobilized to Agrobacteriumtumefaciens by triparental mating. Tomato transformation will beperformed by well established protocols (Tieman, D. et al., Plant Cell4: 667-679 (1992)). Transgenic fruit analysis will be carried out usingdifferent techniques. Determination of S1-TPS1 cDNA integration in theplant genome by genomic Southern gels. Determination of S1-TPS1transcription by Northern blotting and S1-TPS1 protein expression byWestern blotting. Measurement of S1-TPS1 enzyme activity and trehalosecontent will be performed by standard techniques (De Virgilio, C. etal., FEBS Lett. 273: 107-110 (1990) and (Neves, M J. et al., World J.Microbiol. Biotechnol. 10: 17-19 (1994)). Shelf-life is analyzed incontrol and trehalose-producing tomatoes considering several maturationparameters, such as: Softening ratio, ethylene production and fruitquality (texture, colour, sugar content, size) over several weeksperiod.

Example 10 Trehalose-6-Phosphate Assay

1.1 Existing Trehalose-6-Phosphate Assays

To study the importance of Tps1 and more specifically of it's producttrehalose-6-phosphate, it is essential to be able to measure the Tre6Plevels that are effectively present in the cytosol. Until now threemethods have been described to determine Tre6P levels. In a first method(Meleiro et al., 1993, Analytical Biochemistry 213, 171-2) Tre6P wasextracted by a combination of TCA extraction, followed by etherextractions, barium acetate precipitations and anion exchangechromatography. The Tre6P was dephosphorylated by alkaline phosphatase,hydrolysed into two glucose molecules by trehalase and finally theglucose was detected by the glucose oxidase-peroxidase method. Thismethod has been developed to evaluate a procedure to produce and purifylarge quantities of Tre6P. Since the reported Tre6P levels in yeastcells are lower than the glucose, trehalose and Glu6P levels, thismethod would have an enormous background and lack of sensitivity.

A second method uses High Pressure Liquid Chromatography (HPLC) todetect and quantify Tre6P (De Virgilio et al., 1993, Eur J Biochem 212,315-23). With this method it was possible to quantify high Tre6P levelsin tps2Δ mutants after a heat-shock (Reinders et al., 1997, MolMicrobiol 24, 687-95) and detect a transient increase in Tre6P levelsafter the addition of glucose to derepressed wild type yeast cells(Hohmann et al., 1996, Mol Microbiol 20, 981-91). It had a detectionlimit of around 200 μM Tre6P. This is around the steady state levelsthat have been reported in exponentially growing and stationary phasecells (Blazquez et al., 1993, FEBS Lett 329, 51-4). The sensitivity ofthis method does not allow reliable quantification of concentrationspresent in normal yeast strains or strains affected in Tre6P synthesis.

A third method is based on the observation that Tre6P inhibits yeasthexokinase activity in vitro (Blazquez et al., 1994, FEMS Microbiol Lett121, 223-7). The level of this inhibition serves as a measure for theTre6P content of the extracts. Hexokinase activity is measured bydetermining the formation rate of one of its products,fructose-6-phosphate (Fru6P). The intracellular Tre6P in stationary andexponentially growing cells was estimated to be around 200 μM. Theauthors also observed that this inhibitory effect of Tre6P on hexokinaseactivity was suppressed by the presence of glucose. Since in certainconditions, high concentrations of glucose and in general otherinterfering compounds may be present in the extracts, this method isalso not suitable.

1.2 Principles of the Novel Method

The method of the invention has as prime objective to be more sensitiveand more reliable than the existing methods. It is based on the Bacillussubtilis phosphotrehalase enzyme that is encoded by the treA gene. Thisenzyme is functionally related to the bacterial PTS system, andhydrolyses Tre6P into glucose and Glu6P (Helfert et al., 1995, MolMicrobiol 16, 111-120; Rimmele & Boos, 1994, J Bacteriol 176, 5654-64).By measuring the glucose produced in this reaction, the initial amountof Tre6P can be calculated. Glu6P is not used for this purpose becausethis compound is often present in large amounts in yeast cells. It isdifficult to separate it from Tre6P, while the glucose originallypresent in the extracts can easily be separated from the sugarphosphates by anion exchange chromatography (FIG. 16).

1.3 Purification of the B. subtilis Phosphotrehalase Enzyme

The phosphotrehalase enzyme has been purified and characterised by thegroup of M. K. Dahl (Gotsche & Dahl, 1995, J Bacteriol 177, 2721-6;(Helfert, et al., 1995, supra). This group provided the pSG2 plasmidwith the treA gene expressed constitutively behind the strong B.subtilis degO36 promotor.

To obtain high stable expression, the gene was cloned in the pCYB1vector (New England Biolabs) behind the strong IPTG inducible tacpromoter. The treA gene was PCR amplified with the primers CVVS(TGGTGGAT′TA,ATATGAAAACA-GAACAAACGCCATGGTGG) (SEQ ID NO: 13) and CVV6(TTAACA-GCTCTTCC′GCA,AACGATAAACAATGGACTCATATGGGCG) (SEQ ID NO: 14),introducing respectively an AseI and a SapI restriction site at each endof the PCR product and cloned in the pCYB1 plasmid and called pCVV1(FIG. 7). ′and, indicate the site where the restriction enzyme splices.

Comparison of the phosphotrehalase activity of the strains transformedwith the original pSG2 plasmid (EcVV1) or the new pCVV1 (EcVV3) showedthat the latter strain contained 10 times more activity than the strainwith the pSG2 plasmid (FIG. 17).

These EcVV3 cells were grown overnight in 4 ml LB-ampicillin medium. Thenext day this culture was used to inoculate 100 ml LB ampicillin and wasleft to grow again for 3 hours whereafter expression was induced withIPTG (0.5 mM) and the culture incubated for another 3 hours at 37° C.

Cells were centrifuged 10 minutes at 4000 rpm and resuspended in 30 mlbuffer A (25 mM Bis-Tris pH 7, 10 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂). Thecells were broken by passing the resuspended cells twice through aFrench press. The crude extract was then cleared by ultracentrifugationfor 30 minutes at 35000 rpm and the supernatant was passed through a 0,22 μm filter.

The anion exchange chromatography was performed with a linear gradientfrom 100% buffer A to 50% buffer B (25 mM Bis-Tris pH 7, 10 mM KCl, 1 mMCaCl₂, 1 mM MgCl₂, 1M NaCl) in 20 column volumes on a MonoQ HR5/5column. Fractions of 500 μl were collected. The PNPG hydrolysingactivity of each fraction was measured and the two fractions with thehighest activity were pooled and concentrated to 200 μl before thegelfiltration with a Vivaspin column (Vivascience). For thegelfiltration an isocratical elution with 10% buffer B was used on aSuperdex75 column. The two 500 μl fractions with the highest activitywere pooled and concentrated again to 400 μl and stored in aliquots at−30° C. Both columns were run on a AKTA-FPLC system (Pharmacia).

The purification performed in this way gave a 4, 5 fold increase inspecific activity with a recovery of 34%. SDS-PAGE analysis withCoomassie Blue staining showed already a huge overexpression proteinband from the expected size. No other bands were visible in the finalpurified fractions with 10 μg protein per lane.

1.4 Sampling of the Yeast Culture

Samples were taken by spraying 3 ml of a cell suspension into 10 ml of60% methanol at −40° C. This immediately arrests cell metabolism andallows determination of the intracellular metabolites as a function oftime after an experimental manipulation, e.g. adding glucose toderepressed cells (de Koning & van Dam, 1992, Anal Biochem 204, 118-23).

1.5 Extraction of Metabolites

The extraction of metabolites of the frozen cells was performed usingperchloric acid extraction.

1.6 The First Anion Exchange Chromtography

To separate the glucose and trehalose from the Tre6P present in the cellextracts, the weak NH2-SPE anion exchanger (International SorbentTechnology, UK) was selected.

The columns were filled with 500 mg of sorbent resuspended in 3 ml of100% methanol and rinsed with 3 ml 100% methanol. A weak anion was boundto it by equilibrating the column with 0.5 M acetic acid and the excessof acid in the column was rinsed away with 2 times 3 ml of MilliQ water.The sample was applied and the bound sample was rinsed with 3 ml of 20%methanol to eliminate a specific binding. The anions were eluted with a2.5 M ammonia solution pH 12.5. When 250 mg of sorbent was used per 100mg of cells extracted there was no risk of overloading the column. Thesalt did not interfere significantly with the glucose determination. Theeluted extracts were evaporated without loss of Tre6P.

1.7 The Phosphotrehalase Reaction Conditions

Because the enzyme was unstable at the reported pH optimum of 4.5, amore physiological pH of 7 was chosen to perform the incubation. Theincubation buffer was a 50 mM Bis-Tris buffer pH7. The incubationtemperature was 37° C. 2000 U of phosphotrehalase were added and thesamples incubated at 37° C. for 2 hours. One unit was the amount ofenzyme that hydrolysed 1 nmol of Tre6P per minute in the conditionsmentioned.

1.8 The Second Anion Exchange Chromtography

To separate the glucose that was produced in the phosphotrehalasereaction from the other anionic cell compounds a second anion exchangechromatography was performed in the same way as the first one. This timethe flow-through that does not bind to the column and that contains theglucose, was recovered and concentrated by vacuum drying.

1.9 The Glucose Assay

The dried glucose samples were redissolved in 250 μl 50 mM Bis-Trisbuffer pH7. From each sample the glucose concentration was determined in200 μl undiluted sample and in a ⅕ and 1/25 dilution. In a microtiterplate to a sample volume of 200 μl, 20 μl of a reaction mix containing15 units glucose oxidase, 10 units of peroxidase and 100 μgortho-dianisidine was added. This plate was incubated at 37° C. for 45minutes and then the reactions were stopped by adding 40 μl of 56%sulfuric acid. The absorbance was measured at a wavelength of 530 nm.

1.10 Recovery and Reproducibility

The recovery of the perchloric acid extraction procedure is 57%. Therecovery of the complete Tre6P assay is 25%. The detection limit of themethod was 100 μM and Tre6P standard lines are linear in thephysiologically relevant range (FIG. 8). The Tre6P standards were madeby adding known amounts of Tre6P to cells of the tps1Λ strain that lacksthe Tre6P synthase, before the perchloric acid extraction. The standarderror on the slope and intercept of 4 independent standard lines wasrespectively 2 and 9%.

The trehalose-6-phosphate concentrations were measured in yeast strainscontaining the full length and N-terminally deleted plant TPS1 alleles.The strains were either grown till exponential phase or till stationaryphase in either galactose or glucose containing minimal medium. For thisexperiment tps1Δtps2Δ backgrounds were used because the level oftrehalose-6-P in tps1Δ backgrounds is too low to be measured. Thestrains that were used are:

JT6308 1Δ2Δ+pSAL4

PVD44 1Δ2Δ+pSAL4/Sc TPS1

JT6309 1Δ2Δ+pSAL4/S1 TPS1

PVD43 1Δ2Δ+pSAL4/ΔN S1 TPS1

PVD138 1Δ2Δ+PSAL6/At TPS1

JT20050 1Δ2Δ+pSAL6/ΔN At TPS1

PVD150 2Δ+pRS6 (empty plasmid with HIS3 marker)

“1Δ” stands for “tps1Δ”, “2Δ” stands for “tps2Δ”.

After collecting the yeast cells, extracts were made as described inexample 10 and trehalose-6-P concentrations were measured. The resultsare shown in FIG. 18.

There is four times less trehalose-6-phosphate in the extracts preparedfrom the strains containing the plant TPS1 genes compared to the controlstrain overexpressing the yeast TPS1 gene. This lower,trehalose-6-phosphate level might explain why there is stillderegulation of the glucose influx in glycolysis in these strains(example 12). This deregulation of glucose influx was similar forstrains containing either the full length or the N-terminally deletedplant TPS alleles and this fits with the results that we have obtainedhere. There is no difference in trehalose-6-phosphate levels betweenstrains containing the full length or the N-terminally deleted alleles.

Example 11 Chimaeric Fusions of TPS and TPP Domains

To generate a more efficient enzymatic pathway involved in trehalosebiosynthesis a series of chimaeric enzyme fusions were created betweenTPS and TPP domains from TPS1 and TPS2 either from A. thaliana or S.cerevisiae. As shown in FIG. 13, these four fusions consist of: a 1337bp DNA fragment from AtTPS1 (ΔNAtTPS1) encoding a N-terminus truncatedprotein (lacking its first 100 amino acids) of 442 amino acids, obtainedby PCR (94° C., 3 min, 1 cycle; 94° C., 1 min, 52° C., 1 min, 72° C.,1.5 min, 40 cycles; 72° C., 10 min, 1 cycle) using Expand High-fidelityDNA polymerase (Boehringer) with oligonucleotides(5′-CATG-CCATGGCTTAT-AATAGGCAACGACTACT-TGTAGTG-3′ (SEQ ID NO: 6),underlined NcoI site and bold initiation codon) and(5′-CGGGATCCAGCTGTCATGTTTAGGGC-TTGTCC-3′ (SEQ ID NO: 15), underlinedBamHI site), fused to a 1138 bp DNA fragment from AtTPPB encoding thefull-length protein of 374 amino acids obtained by PCR (94° C., 3 min, 1cycle; 94° C., 1 min, 52° C., 1 min, 72° C., 1.5 min, 40 cycles; 72° C.,10 min, 1 cycle) using Expand High-fidelity DNA polymerase (Boehringer)with oligonucleotides (5′-CGGGATCCACTA-ACCAGAATGTCATCG-3′ (SEQ ID NO:16), underlined BamHI site) and (5′-GGGGTACCTCACTCTT-CTCCCACTGTCTTCC-3′(SEQ ID NO: 17), underlined KpnI site and bold stop codon).

A second fusion consists of ΔNAtTPS1 fused to a 1358 bp DNA fragmentfrom ScTPS2 encoding 397 amino acids from its C-terminus, obtained byPCR (94° C., 3 min, 1 cycle; 94° C., 1 min, 50° C., 1 min, 72° C., 1.5min, 40 cycles; 72° C., 10 min, 1 cycle) using Expand High-fidelity DNApolymerase (Boehringer) with oligonucleotides(5′-CGGGATCCGCTAAATCT-ATTAACATGG-3′ (SEQ ID NO: 18), underlined BamHIsite) and (5′-CGGGGTACCATGG-TGGGTTGAGAC-3′ (SEQ ID NO: 19), underlinedKpnI site).

A third construct led to a fusion between a 1531 bp DNA fragment fromScTPS1 encoding for a 492 amino acid protein, obtained by PCR (94° C., 3min, 1 cycle; 94° C., 1 min, 52° C., 1 min, 72° C., 1.5 min, 40 cycles;72° C., 10 min, 1 cycle) using Expand High-fidelity DNA polymerase(Boehringer) with oligonucleotides (5′-CCGCTCGAGGGTACTC-ACATACAGAC-3′(SEQ ID NO: 20), underlined XhoI site) and(5′-CGGGATCCGGTGGCA-GAGGAGCTTGTTGAGC-3′ (SEQ ID NO: 21), underlinedBamHI site), and AtTTPB.

The last fusion was made between ScTPS1 and ScTPS2 DNA fragmentsobtained as described above.

The PCR fragments were digested with appropriate restriction enzymes(FIG. 13) and subcloned in pRS6 vector. Yeast tps1Δ, tps2Δ andtps1Δtps2Δ mutant strains were transformed and selected in SDGal (-his).Complementation was assayed in SDGlc (-his minimal medium) and growth at38.3° C.

Example 12 Expression in Yeast Tpsli Strains, of N-Terminally DeletedPlant Tps1 Genes, Restores the Growth on Glucose but does not SuppressHyperaccumulation of Sugar Phosphates

Deletion of TPS1 in S. cerevisiae results in a pleiotropic phenotype(Van Aelst et al., Mol. Microbiol., 8, 927-943, 1993). One of thephenotypes is that such a strain can not grow anymore on rapidlyfermentable sugars. Why tps1Δ strains can not grow on glucose is not yetclarified. Three different hypotheses have been proposed (Hohmann andThevelein, Trends in Biol. Sciences, 20, 3-10, 1995). When tps1Δ strainsare shifted from a glycerol to glucose containing medium there is arapid accumulation of sugar phosphates and a rapid drop in ATPconcentration. Apparently the TPS1 gene has a function in the control ofglucose influx into glycolysis. Because transport and phosphorylationare coupled, all the sugar that is coming into the cell isphosphorylated. Reducing the Hxk2 activity can suppress the growthdefect phenotype of tps1Δ strains on glucose and fructose (Hohmann etal., Current genetics 23, 281-289, 1993). In vitro studies have clearlyindicated that the product of the Tps1 protein, trehalose-6-phosphate,inhibits the activity of Hxk2 and as such, could control the flux ofglucose into glycolysis.

When the S. lepidophylla homologue of TPS1 was expressed in yeast, aclear difference is seen in growth on glucose containing medium betweenthe full length clone and the N-terminal deletion construct. Tps1Δstrains transformed with the full length S1 TPS1 under the control ofthe CUP1 promoter do not grow on glucose. Expression in a tps1Δ strainof a construct where the first 300 bp encoding the first 100 amino acidsof the S1 Tps1 protein are deleted results in growth on glucose. Soapparently, the full-length clone can not solve the glucose influxproblem, whereas the N-terminal deletion is able to control the glucoseinflux.

To test this, an experiment was performed where the concentration of thefirst metabolites in glycolysis was measured. The following strains wereused:

PVD72 Tps1Δ+pSAL4

PVD14 Tps1Δ+pSAL4/Sc TPS1

PVD73 Tps1Δ+pSAL4/S1 TPS1

PVD15 Tps1Δ+pSAL4/ΔN S1 TPS1

For the determination of the glycolytic metabolites, cells were grown onSDglycerol medium till exponential phase. Cells were harvested bycentrifugation. The pellet was washed once, resuspended and thenincubated in YP medium at 30° C. Glucose was added to a finalconcentration of 100 mM and CuSO₄ was added to a final concentration of100 μM.

Before and after the addition of glucose, samples were taken at the timeintervals indicated and immediately quenched in 60% methanol at −40° C.

Determination of the glycolytic metabolites was performed on thesesamples essentially as described by de Koning and van Dam (Anal.Biochem. 204, 118-123, 1992). Using the total amount of protein in thesample as determined according to Lowry et al (J. Biol. Chem. 193,265-275, 1951), and the assumption of a yeast cytosolic volume of 12 μlper mg protein, cytosolic concentrations were calculated in mM. FIG. 14shows the result of a representative experiment.

The results clearly indicate that in this short period after theaddition of glucose, there is no difference between the metaboliteconcentrations of the full length and the N-terminal deleted S.lepidophylla TPS1 gene. There is a clear hyper accumulation of sugarphosphates after the addition of glucose what is similar to what can beseen for the tps1Δ strain.

These results with the N-terminal deletion constructs show that theaccumulation of sugar phosphates is not related to the fact that yeastcells can or cannot grow on glucose. This means that the N-terminal partis important for the control of glucose influx into glycolysis. It alsoimplies that a tps1Δ strain containing the ΔN S1 or ΔN At TPS1 genes canbe used as a tool to increase the total flux through glycolysis. Thisstrain grows perfectly on glucose but still a hyper accumulation ofmetabolites in the upper part of glycolysis is seen. Overexpression ofthe enzymes downstream in glycolysis results in a higher flux.

Since the metabolite concentration was only measured over a short periodof time after the addition of glucose, another experiment was done wherethe metabolite concentration was measured during exponential growth andduring stationary phase. These results are shown in FIG. 15.

These data confirm the results obtained in the first experiment. Thereis a hyperaccumulation of sugar phosphates in the tps1Δ straintransformed with the N-terminal deletion construct. From FIGS. 14 and 15it follows that the difference between the tps1Δ strain and the tps1Δstrain containing the ΔN S1 TPS1 expression plasmid is the ATP level.Whereas the ATP level in the tps1Δ strain drops to zero this is not thecase in the other strains. The ATP level that is left is apparentlyenough to grow on glucose.

The invention claimed is:
 1. A method of delaying growth of a plantcomprising the steps of: a) providing a TPS protein encoded by a plantTPS gene; b) designing a suitable modification to the plant TPS gene bytruncating or deleting an N-terminal part of the TPS protein encoded bythe plant TPS gene in order to achieve an increasedtrehalose-6-phosphate synthase activity; c) cloning the thus modifiedgene into an expression vector under the control of a constitutive,inducible and/or organ-specific promoter; and d) transforming the plantor tissue from the plant with the thus obtained expression vector,wherein the growth of the plant is delayed, and wherein the TPS gene isselected from the group consisting of rice, Arabidopsis thaliana,Selaginella lepidophylla, apple, sugar beet, sunflower (Helianthusannuus), tobacco (Nicotiana tabacum), and soybean (Glycine max).
 2. Amethod of delaying growth of a fungus comprising the steps of: a)providing a TPS protein encoded by a plant TPS gene; b) designing asuitable modification to the plant TPS gene by truncating or deleting anN-terminal part of the TPS protein encoded by the plant TPS gene inorder to achieve an increased trehalose-6-phosphate synthase activity;c) cloning the thus modified gene into an expression vector under thecontrol of a constitutive, inducible and/or organ-specific promoter; andd) transforming the fungus with the thus obtained expression vector,wherein the growth of the fungus is delayed.
 3. The method as claimed inclaim 2, wherein the fungus is selected from the group consisting ofAspergillus niger, Agaricus bisporus, Pictia pastoria, Kluyveromyceslactis and methylotrophic yeasts.
 4. A fungus having an improvedfermentation capacity comprising a TPS gene, wherein the TPS gene istruncated at an N-terminal part of a TPS protein encoded by the TPS genein order to achieve an increased trehalose-6-phosphate synthaseactivity.
 5. A transgenic plant comprising a nucleotide sequenceselected from the group consisting of: ΔN At TPS1 fused with At TPPB; ΔNAt TPS1 fused with Sc TPS2; and Sc TPS1 fused with At TPPB.